Metabolic and Transcriptomic Adaptation of Lactococcus lactis subsp. lactis Biovar diacetylactis in Response to Autoacidification and Temperature Downshift in Skim Milk

Abstract

For the first time, a combined genome-wide transcriptome and metabolic analysis was performed with a dairy Lactococcus lactis subsp. lactis biovar diacetylactis strain under dynamic conditions similar to the conditions encountered during the cheese-making process. A culture was grown in skim milk in an anaerobic environment without pH regulation and with a controlled temperature downshift. Fermentation kinetics, as well as central metabolism enzyme activities, were determined throughout the culture. Based on the enzymatic analysis, a type of glycolytic control was postulated, which was shared by most of the enzymes during the growth phase; in particular, the phosphofructokinase and some enzymes of the phosphoglycerate pathway during the postacidification phase were implicated. These conclusions were reinforced by whole-genome transcriptomic data. First, limited enzyme activities relative to the carbon flux were measured for most of the glycolytic enzymes; second, transcripts and enzyme activities exhibited similar changes during the culture; and third, genes involved in alternative metabolic pathways derived from some glycolytic metabolites were induced just upstream of the postulated glycolytic bottlenecks, as a consequence of accumulation of these metabolites. Other transcriptional responses to autoacidification and a decrease in temperature were induced at the end of the growth phase and were partially maintained during the stationary phase. If specific responses to acid and cold stresses were identified, this exhaustive analysis also enabled induction of unexpected pathways to be shown.Lactic acid bacteria (LAB) are gram-positive microorganisms that have a real economic impact due to their use in many different food transformation processes. The bacterium Lactococcus lactis, which is commonly used as a starter bacterium in the manufacture of different dairy products, such as cheese, butter, and buttermilk, is generally considered a model for metabolism regulation, physiology, and genetic studies in LAB. In industrial conditions, as well as in natural ecological niches (plants, animals, and gastrointestinal tracts), different physicochemical stresses (including acid, thermic, oxidative, and osmotic stresses) are encountered, leading to suboptimal growth. Acid stress turns out to be the major stress that L. lactis is confronted with during fermentative processes. L. lactis, which is generally considered a homofermentative bacterium, is able to convert more than 90% of the sugar metabolized into lactic acid, whose accumulation is responsible for the progressive acidification of the medium, which leads to autoinhibition of lactococcal growth. This process, which is generally attributed to bacterial membrane permeability to the nondissociated form of lactic acid, increases when the medium is acidified and results in a decrease in the internal pH (12). This cytoplasmic acidification is known to be responsible for growth inhibition (11, 21) or a loss of cell viability (10,14,26,30) due to limitation of the activity...

Impact of Aeration and Heme-Activated Respiration on Lactococcus lactis Gene Expression: Identification of a Heme-Responsive Operon

“…Enzymes encoded by these genes reportedly contribute to raising internal pH in acidification conditions by liberation of NH 3 and are induced by acid stress (8), (47). These genes are reportedly downregulated in the presence of O 2 in different bacterial species (e.g., see references 16 and 26).…”

Transcriptome Analysis of Lactococcus lactis in Coculture with Saccharomyces cerevisiae

“…It is well known that the diminished growth rate, whatever the cause of the slowing down, has large implications for global gene expression, affecting functions such as amino acid transporters, sugar metabolism, large and small ribosomal subunits, etc. (30,31). On the other hand, it is expected that very few genes will show variation of expression during exponential growth phases in which specific rates are comparable.…”

“…lactis is considered a model lactic acid bacterium, encountered extensively in numerous food fermentation processes, particularly in cheese production, industrial processes, and natural ecosystems. DNA arrays for L. lactis strain IL-1403, the first lactococcal strain to be sequenced (6), have recently been used for transcriptome analysis of L. lactis pure cultures (4,19,30,31). However, in most cases, L. lactis lives together with multiple microorganisms such as lactobacilli, corynebacteria, and yeasts (7,11,24,26,28,33,35).…”

Unraveling Microbial Interactions in Food Fermentations: from Classical to Genomics Approaches

“…A number of studies have appeared that describe successful transcriptome or proteome analyses on samples from fermented substrates and even from highly complex materials such as fecal samples (81,178). Most studies of dairy fermentations use skim milk, in which the precipitation of casein may be prevented by pretreatment with sodium citrate (126,139). These studies have revealed several previously undescribed metabolic adaptations upon growth in milk, such as the induction of pyruvate-formate lyase in S. thermophilus that may serve as the supply of formate required for the biosynthesis of purine bases or other anabolic processes (37) (Fig.…”